Experiments carried out in our laboratory have established that all the carbon and nitrogen atoms of the 5,6-dimethylbenzimidazole (DBI) moiety of vitamin B12 may be derived from 6,7-dimethyl-8-ribityllumazine (lumazine) precursors. We now propose to investigate the details of the enzymatically catalyzed transformations which comprise the biosynthetic pathway from lumazine to DBI in Propionibacteria. Affinity columns containing covalently bound analogues of the potential intermediates of this biosynthetic pathway will be prepared and used to detect and to concentrate the biosynthetic enzymes present in Propionibacteria cell-free extracts. Specifically labeled substrates will then be prepared and used to assay, to purify, and finally to characterize the enzymatic activities which are involved in DBI biosynthesis in Propionibacteria. In addition, we propose to investigate the nature of the four carbon biosynthetic unit involved in the biochemical formation of lumazine from a ring opened, guanosine derivative. It has been proposed that this key four carbon biosynthetic unit is derived from pentose cycle metabolism. Because this four carbon biosynthetic unit ultimately is incorporated into the DBI moiety of B12, the DBI labeling patterns resulting from a series of substrates may be used to determine the nature of this four carbon biosynthetic unit.